Restoration Of miR-200c Research Articles

Abstract P2-01-04: Epithelial-mesenchymal transition promotes triple negative breast cancer immune suppression

Abstract Background: Epithelial-Mesenchymal Transition (EMT) is a developmental process co-opted by carcinomas to facilitate metastasis. Triple negative breast cancer (TNBC), which has often undergone at least partial EMT, has a poor prognosis due to frequent and rapid recurrence as metastatic disease when compared to other breast cancer subtypes. To identify drivers of TNBC progression microRNA miR-200c was restored to the human TNBC mesenchymal-like BT549 cell line, which decreased 80% of the pan-EMT mesenchymal signature and restored 60% of the epithelial signature. In addition to decreasing proteins involved in immune-suppression via contact-dependent mechanisms, such as CD274 (PD-L1) and (CD273) PD-L2, Ingenuity Pathway and GSEA analysis revealed reduced expression of genes encoding enzymes, including Tryptophan 2,3-Dioxygenase (TDO2) and Heme Oxygenase-1 (HO-1), that generate immune-suppressive metabolites that function in a non-contact dependent manner. TNBC utilizes these and other immune-suppressive factors made by fetal trophoblasts to suppress the maternal immune system to achieve fetal tolerance during pregnancy, suggesting that TNBC may mimic a physiologically relevant mechanism to promote immune evasion. Hypothesis: TNBC metabolism is altered as part of an EMT program resulting in generation of immune-suppressive metabolites that promote metastasis by facilitating contact-independent immune evasion. Methods: Expression and activity of TDO2 and HO-1 were determined by UHPLC-MS for kynurenine (generated by TDO2) and bilirubin (generated by HO-1) in multiple TNBC cell lines. Single cell RNAseq was performed to identify genes co-expressed with these enzymes. To develop an immune competent preclinical model, doxycycline inducible miR-200c (TripZ-200c) was stably introduced into the Met-1 mammary carcinoma cell line derived from the MMTV-PyMT mouse model developed in FVB/NJ mice. Results were confirmed via transient miR-200c expression in 66cl-4 mammary carcinoma cells (from a spontaneous BALB/c tumor). Results: Restoration of miR-200c decreased TDO2 and HMOX1 (HO-1) by 80% and 40% respectively in Met-1 and 66cl-4 mammary carcinoma cells and reduced secretion of kynurenine and bilirubin. HO-1 was increased in lung metastasis from Met-1 and 66cl-4 cells when compared to cells prior to tail vein injection. ScRNAseq revealed specific gene expression signatures co-expressed with TDO2 and are being analyzed to better detect TDO2 in tumor tissues. In vivo studies are underway to investigate the effects of inhibiting tumor TDO2 and HO-1 on infiltrating lymphocyte composition, the anti-tumor immune response, and tumor progression. Conclusions: Restoration of miR-200c revealed EMT-associated alterations in tumor metabolism and consequent secretion of immune-suppressive metabolites produced by TDO2 and HO-1. We propose that targeting these contact-independent immune-suppressive factors may complement current contact-dependent therapeutic strategies (checkpoint inhibitors) to boost anti-tumor immune cell function and thereby prevent or decrease TNBC metastasis. Citation Format: Williams MM, Christenson JL, O'Neill K, Rogers TJ, Greene LI, Slansky J, Richer JK. Epithelial-mesenchymal transition promotes triple negative breast cancer immune suppression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-04.

Abstract A18: miR-9 and miR-200 regulate PDGFRβ-mediated endothelial differentiation of neoplastic cells in triple-negative breast cancer

Abstract Tumor vascularization is a fundamental step in solid tumor progression and is orchestrated by different pathways of vasculogenesis. In malignant tumors, neoplastic cells can differentiate into endothelial-like cells acquiring the expression of endothelial markers (i.e. CD31 and CD34) and participating in the formation of vascular-like structures that functionally deliver oxygen and nutrients to the tumor site. We recently identified PDGFRβ as an important player of this process in triple negative breast cancer (TNBC). Interestingly, increasing evidence supported a connection between PDGFRβ and epithelial to mesenchymal transition (EMT), important step for the endothelial trans-differentiation process. PDGFR signaling arose as a promising target for TNBC; thus a better understanding of this new role of PDGFRβ could be relevant in the biology and treatment of TNBC. We then aimed at investigating microRNAs able to regulate tumor vascularization via PDGFRβ-mediated endothelial differentiation in TNBC, focusing on miR-9 and miR-200 family in light of their known relation with PDGFRβ and EMT. Tube formation assay showed that in MDA-MB-231 and MDA-MB-157 TNBC cell lines transfection of miR-9 and miR-200 family members (miR-200b and miR-200c) promoted or inhibited, respectively, the ability of cells to form vascular-like structures when seeded on matrigel. Stimulation of PDGFRβ with PDGF-BB ligand induced miR-9 expression and enhanced the loop formation ability of treated cells. This advantage was, however, almost completely abrogated by the concomitant inhibition of miR-9, thus demonstrating that miR-9 contributed to PDGFRβ-mediated vasculogenic properties of TNBC. Moreover, we found that silencing of STARD13, direct target of miR-9 validated by luciferase reporter assay, improved the vasculogenic potential. Ectopic expression of miR-200 members, instead, suppressed PDGFRβ at protein level in both TNBC cell lines. Furthermore, the silencing of ZEB1, known target of miR-200 family, induced downregulation of PDGFRβ at protein and mRNA level; interestingly, the mRNA levels of ZEB1 and PDGFRβ strongly correlated in the TNBC subset of the TGCA dataset. The in vivo effect of miR-9 and miR-200 family on vasculogenic properties of TNBC was then validated first through the generation of MDA-MB-231 clones for stable inhibition of miR-9 and restoration of miR-200c; the resulting miR-9 sponge and miR-200c xenografted tumors showed lower number of vascular lacunae than controls, identified through immunohistochemical (IHC) staining for CD31. These results were validated in orthotopic MDA-MB-231 breast tumors treated with miR-9 inhibitors or miR-200c mimics by peritumoral injection. We finally evaluated PDGFRβ, by IHC, and miR-9 and miR-200c, by Real-time PCR, in a cohort of TNBC. IHC analysis demonstrated that PDGFRβ staining identified tumor cells participating in vascular lacunae, data further confirmed by immunofluorescence co-localization of PDGFRβ and CD31. Interestingly, unlike miR-9, the expression of miR-200c negatively associated with both the presence of vascular lacunae and tumor nests positive for PDGFRβ. Finally, in situ hybridization analysis revealed that miR-200c was mainly expressed by tumor cells. In conclusion, our results suggest that miR-9 and miR-200 family play an opposite role in the regulation of the vasculogenic aptitude of TNBC. MiR-9 is induced by PDGFRβ and enhances the vasculogenic properties of tumor cells in part through the negative regulation of STARD13. MiR-200, instead, inhibits this aggressive phenotype indirectly suppressing PDGFRβ through targeting of ZEB1. Citation Format: Elvira D'Ippolito, Ilaria Plantamura, Lucia Bongiovanni, Patrizia Casalini, Sara Baroni, Claudia Piovan, Rosaria Orlandi, Ambra V. Gualeni, Annunziata Gloghini, Anna Rossini, Filippo De Braud, Elda Tagliabue, Claudio Tripodo, Marilena V. Iorio. miR-9 and miR-200 regulate PDGFRβ-mediated endothelial differentiation of neoplastic cells in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A18.

MiR‐200c suppresses TGF‐β signaling and counteracts trastuzumab resistance and metastasis by targeting ZNF217 and ZEB1 in breast cancer

Resistance to trastuzumab and concomitantly distal metastasis are leading causes of mortality in HER2-positive breast cancers, the molecular basis of which remains largely unknown. Here, we generated trastuzumab-resistant breast cancer cells with increased tumorigenicity and invasiveness compared with parental cells, and observed robust epithelial-mesenchymal transition (EMT) and consistently elevated TGF-β signaling in these cells. MiR-200c, which was the most significantly downregulated miRNA in trastuzumab-resistant cells, restored trastuzumab sensitivity and suppressed invasion of breast cancer cells by concurrently targeting ZNF217, a transcriptional activator of TGF-β, and ZEB1, a known mediator of TGF-β signaling. Given the reported backward inhibition of miR-200c by ZEB1, ZNF217 also exerts a feedback suppression of miR-200c via TGF-β/ZEB1 signaling. Restoration of miR-200c, silencing of ZEB1 or ZNF217 or blockade of TGF-β signaling increased trastuzumab sensitivity and suppressed invasiveness of breast cancer cells. Therefore, our study unraveled nested regulatory circuits of miR-200c/ZEB1 and miR-200c/ZNF217/TGF-β/ZEB1 in synergistically promoting trastuzumab resistance and metastasis of breast cancer cells. These findings provide novel insights into the common role of EMT and related molecular machinery in mediating the malignant phenotypes of breast cancers.

Abstract B70: Decreasing ovarian cancer metastases and progression via restoration of microRNAs that target mediators of anoikis- and chemoresistance.

Abstract Background: Epithelial ovarian cancer (EOC) accounts for the majority of deaths from gynecological malignancies, with a 5-year survival rate for women with advanced stage disease of only 46%. In EOC metastasis primarily occurs by a mechanism termed direct seeding, which involves shedding of tumor cells from the primary site into the peritoneal cavity. To allow survival in suspension before attachment at metastatic sites, ovarian cancer cells must acquire resistance to anoikis, a form of apoptosis initiated by detachment from native extracellular matrix. A targeted therapy that could simultaneously affect multiple factors critical for ovarian cancer progression such as anoikis resistance, chemoresistance and attachment to metastatic sites in the peritoneal cavity, would be a revolutionary breakthrough for this aggressive malignancy. Due to their ability to inhibit multiple targets involved in tumor survival, microRNAs (miRNAs) are attractive therapeutic candidates. We previously demonstrated that miR-200c is downregulated in ovarian cancer cell lines and restoration of miR-200c increased sensitivity to taxanes in vitro, by targeting TUBB3, a known mediator of chemoresistance. We tested the effect of miR-200c restoration in an intraperitoneal xenograft model of human ovarian cancer and observed dramatically decreased tumor formation. Further, even in established tumors, inducible restoration of miR-200c alone and in combination with paclitaxel, resulted in significantly reduced tumor burden. Hypothesis: Since restoration of miR-200c to multiple anoikis resistant EOC cell lines increased anoikis sensitivity and reduced adherence to biological substrates in vitro, we hypothesized that the decreased intraperitoneal tumor burden observed upon miR-200c restoration resulted from increased anoikis sensitivity. Methods and results: To elucidate mechanisms of anoikis resistance in ovarian cancer and identify targets involved in the ability of miR-200c to reverse anoikis resistance, we performed gene and miRNA expression profiling of attached Hey ovarian cancer cells as compared to the same cells grown under conditions of forced suspension for 24h. We identified miR-193 as downregulated in anoikis resistant cells in suspension. Among numerous genes predicted as miR-200c and/or miR-193 targets, SCLC7A11 (xCT), which encodes the light chain of the cystine/glutamate antiporter system xc, was increased by 10.7 fold in suspended versus attached cells. The xc- system is involved in glutathione (GSH) synthesis, mediating intracellular defense against oxidative stress. Reactive oxygen species (ROS) increased in anoikis-sensitive (OvCA 433) cells in suspension, but not in anoikis-resistant cells (Hey), suggesting that xCT might protect these cells from ROS-induced cell death. Consistent with this hypothesis, xCT mRNA and protein increased in anoikis resistant EOC cells in suspension, and transient xCT knockdown rendered cells anoikis sensitive. Interestingly, the xCT 3'UTR contains three predicted binding sites for miR-200c and two sites for miR-193b, and restoration of either miRNA blocked the suspension induced-increase in cXT protein, suggesting that both miRNAs target this gene. Conclusions: Our results indicate that the ability of miR-200c to reduce tumor burden results, at least in part, from its ability to restore anoikis sensitivity by inhibiting a suspension-induced increase in xCT that protects tumor cells from ROS. Thus, miR-200c could be a powerful candidate to block progression of ovarian cancer by enhancing anoikis sensitivity and restoring chemo-sensitivity to already attached tumor. Citation Format: Diana M. Cittelly, Dawn R. Cochrane, Erin N. Howe, Irina Dimitrova, Miriam D. Post, Broaddus R. Russell, Monique A. Spillman, Jennifer K. Richer. Decreasing ovarian cancer metastases and progression via restoration of microRNAs that target mediators of anoikis- and chemoresistance.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B70.

Down-regulation of microRNA-200c is associated with drug resistance in human breast cancer

Drug resistance remains a major clinical obstacle to successful treatment in breast cancer patients, and the evidence of microRNAs involvement in cancer drug resistance has been emerging recently. However, the role of microRNA-200c (miR-200c) in modulating chemoresistance of breast cancer remains largely unexplored. Here, we investigated the miR-200c expression in tumor specimens obtained from thirty-nine breast cancer patients who received neoadjuvent chemotherapy by quantitative real-time PCR. Down-regulated miR-200c was observed in non-responders as compared to responders. In addition, miR-200c expression was observed to be down-regulated over 800-fold in human breast cancer cells resistant to doxorubicin MCF-7/ADR as compared to the parental MCF-7 cells. Up-regulation of miR-200c with transfection of miR-200c mimics in breast cancer cells could enhance the chemosensitivity to epirubicin and reduce expression of multidrug resistance 1 mRNA and P-glycoprotein. Moreover, our study demonstrated that restoration of miR-200c in MCF-7/ADR cells could increase intracellular doxorubicin accumulation determined by flow cytometry. Taken together, our findings suggest that miR-200c may act as a promising therapeutic target for improvement of responsiveness to chemotherapy in breast cancer.

Abstract 137: MicroRNA-200c restores anoikis sensitivity in breast and gynecological cancers

Abstract Background: MicroRNA-200c directly targets ZEB1, an epithelial to mesenchymal (EMT)-inducing transcriptional repressor of E-cadherin and other key determinates of epithelial identity and polarity. Therefore, restoration of miR-200c results in repression of ZEB1, dramatic reduction in migration and invasion, and increased sensitivity to microtubule targeting chemotherapeutics. We hypothesized that in addition to ZEB1, miR-200c targets additional genes that contribute to its myriad effects on maintaining the epithelial phenotype by repressing mesenchymal genes. Results: We performed microarray analysis following restoration of miR-200c to aggressive endometrial cancer cells and identified 18 genes that were both significantly repressed and predicted to be direct targets of miR-200c. These genes, including fibronectin 1 (FN1), class III beta-tubulin (TUBB3), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), moesin (MSN1) and Rho GTPase activating protein 19 (ARHGAP19), are all typically expressed only in non-epithelial cells, such as fibroblasts or neurons. Utilizing luciferase reporter assays and mutational analysis, we validate each of these genes as direct targets of miR-200c. We also demonstrate that restoration of miR-200c leads to a dramatic decrease in mRNA and protein levels of these targets. Further we find that restoration of miR-200c restores anoikis sensitivity to suspended cells, leading to over a 1.5 fold increase in cell death. TrkB is a potent suppressor of anoikis in breast and ovarian cancer models. Addition of TrkB lacking the miR-200c binding site reversed the ability of miR-200c to suppress anoikis. Interestingly, neurotrophin 3 (NTF3), a ligand for TrkB, contains 2 putative miR-200c binding sites and is down regulated upon restoration of miR-200c. Thus, loss of miR-200c in carcinoma cells allows inappropriate translation of both TrkB and NTF3, setting up an autocrine loop that results in anoikis resistance. Conclusions: Our data demonstrate that miR-200c serves as a “guardian of the epithelial phenotype” and suppressor of EMT by directly targeting and repressing a program of genes that should not be on in differentiated epithelial cells. We identify a novel function of miR-200c, the ability to suppress anoikis resistance, an important yet understudied step in the metastatic cascade. Funding source: DOD Breast Cancer Program Idea Award BC084162 to JKR Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 137. doi:10.1158/1538-7445.AM2011-137

Abstract 498: MicroRNA200c attenuated metastatic abilities in head and neck squamous cell carcinoma-derived cancer cells

Abstract MicroRNA-200c (miR200c) is emerging as an important regulator of epithelial-mesenchymal transition (EMT), tumorigenicity and cancer metastasis. Recent investigation showed miR200c can regulate the stemness in breast cancer stem cell through directly binding the 3’UTR of Bmi1. However, the role of miR200c in head and neck squamous cell carcinoma (HNSCC) and HNSCC-associated CSC has never been determined. In this study, we first demonstrated that miR200c expression was significantly decreased but Bmi1 expression was increased in HNSCC tissues as compared to adjacent normal cells. Secondly, HNSCC-ALDH1+CD44+ cells presented tumor initiating and radioresistant properties with high EMT. Consistently, the Bmi1 expression was significantly up-regulated and miR200c was down-regulated in HNSCC-ALDH1+CD44+ compared to other isolated subsets of HNSCC cells. Furthermore, miR-200c overexpression or Bmi1 knockdown through lentiviral infection could significantly inhibit the clonogenic and invasive abilities of HNSCC-ALDH1+CD44+ cells. Moreover, overexpression of Bmi-1 promoted HNSCC-ALDH1−CD44− to acquire CSC properties which are similar to HNSCC-ALDH1+CD44+. In contrast, overexpressing of miR200c shifted transcriptomes profiles of ALDH1+CD44+ similar to those of ALDH1-/CD44- populations and abolished self-renewal as well as EMT properties in ALDH1+CD44+. Most importantly, overexpression of miR-200c in ALDH1+CD44+ or ALDH1−CD44−Bmi-1over cells effectively blocked tumor growth and lung metastasis, and significantly prolonged survival time in neck orthotropic-transplanted immunocompromised mice. Lastly, miR200c is significantly reduced in high grade, metastatic HNSCC tissues compared to low-grade HNSCC tissues. In sum, these results show that miR200c negatively modulates Bmi1 expression and plays a role as a tumor suppressor in HNSCC through eliminating HNSCC-CSC population. Restoration of miR200c in HNSCC and CSC may pave a new approach for therapeutic treatment in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 498. doi:10.1158/1538-7445.AM2011-498

MicroRNA‐200c attenuates tumour growth and metastasis of presumptive head and neck squamous cell carcinoma stem cells

MicroRNA-200c (miR200c) is emerging as an important regulator of tumourigenicity and cancer metastasis with a strong capacity for inducing epithelial-mesenchymal transitions. However, the role of miR200c in head and neck squamous cell carcinoma (HNSCC) and HNSCC-associated cancer stem cells (HNSCC-CSCs) is unknown. In this study, the expression of miR200c in the regional metastatic lymph node of HNSCC tissues was significantly decreased, but BMI1 expression was increased as compared to parental tumours. Importantly, site-directed mutagenesis with a luciferase reporter assay showed that miR200c targeted the 3' UTR of BMI1 in HNSCC cells. Isolated HNSCC-derived ALDH1(+) /CD44(+) cells displayed CSC-like tumour initiating and radio-resistant properties. The expression levels of miR200c were significantly down-regulated while BMI1 was increased in HNSCC-ALDH1(+) /CD44(+) compared to the other subsets of HNSCC cells. Furthermore, increased miR200c expression or knockdown of BMI1 could significantly inhibit the malignant CSC-like properties of ALDH1(+) /CD44(+) cells. miR200c over-expression further down-regulated the expressions of ZEB1, Snail and N-cadherin, but up-regulated E-cadherin expression in ALDH1(+) /CD44(+) cells. Finally, a xenotransplantion study confirmed that over-expression of miR200c or BMI1 knockdown effectively inhibited the lung metastatic ability and prolonged the survival rate of ALDH1(+) /CD44(+) -transplanted mice. In summary, miR200c negatively modulates the expression of BMI1 but also significantly inhibits the metastatic capability of epithelial-mesenchymal transitions in malignant HNSCC by reducing the expression of BMI1/ZEB1. Restoration of miR200c in HNSCC and CSCs may be a promising therapeutic approach.

Abstract 2063: Reversal of chemoresistance to Paclitaxel by reintroduction of microRNA-200c in ovarian cancer cells

Abstract MircoRNAs control the post-translational regulation of many genes involved in tumorigenesis including those involved in epithelial to mesenchymal transition (EMT), cellular migration, adhesion, invasion and sensitivity to chemotherapy. The transcription factor ZEB1, which represses E-cadherin and other genes involved in maintaining polarity, is normally not expressed in epithelial cells. However, it is inappropriately expressed in high-grade carcinomas, which lack microRNA −200c (miR-200c). Class III beta-tubulin (TUBB3) is abnormally expressed in carcinomas resistant to microtubule-targeting chemotherapies. We have demonstrated previously that miR-200c, in addition to directly targeting ZEB1/2, also directly targets TUBB3 (as well as other genes typically only expressed in mesenchymal or neuronal cells) resulting in increased sensitivity to paclitaxel in breast and endometrial cancer. TUBB3 has been found to be overexpressed in ovarian cancers resistant to We hypothesized that restoration of miRNA-200c would enhance ovarian cancer chemosensitivity to paclitaxel. Results: Restoration of miR-200c into Hey ovarian cancer cells using microRNA mimic reduced in vitro migration and invasion by 84 and 86% respectively. Restoration of miR-200c also restored chemosensitivity to paclitaxel in cell death assays (85% increase in cell death) and reduced cell survival in clonogenic assays (49-55% decrease in total colony area and 67-70% decrease in the number of colonies). We proved that this was a direct result of miR-200c targeting of TUBB3 since introduction of exogenous TUBB3 expression vector lacking the miR-200c target site inhibited the miR-200c-mediated increase in sensitivity to paclitaxel. In addition, ovarian cancer cells transiently transfected with miR-200c mimic demonstrate statistically significantly decrease in adhesion to basement membrane complex and its components. We are currently in the process of analyzing 20 patient samples of high-grade serous carcinoma for expression of ZEB1 and TUBB3 by IHC and miR-200c by in-situ hybridization and correlating this data with clinical course and response to chemotherapy. In addition we have created Hey ovarian cancer cells expressing miR-200c in a constitutive manner and are evaluating the effect of restoration of miR-200c on intraperitoneal tumor burden and chemoresistance in a pre-clinical mouse model of ovarian cancer. Conclusions: Re-introduction of miR-200c restores chemosensitivity to paclitaxel through down-regulation of TUBB3 in Hey ovarian cancer cells. Because restoration of miR-200c is crucial to maintenance of epithelial identity, reduces adherence to various substrates, and increases sensitivity to paclitaxel, it is holds potential as a therapeutic strategy for chemoresistant ovarian cancers. Funded by the Colorado Cancer League grant to JKR and ID. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2063.